Chemically modified nucleotides have been extensively used in the study of many complicated biological systems. In particular, they have proven indispensable in the analysis of protein-nucleic acid interactions, the determination of genotypes, and the sequencing of nucleic acids. In general, these applications rely on differences in the chemical reactivity or electronic properties of the modified nucleotides as compared to the naturally occurring counterpart. Analogs of nucleotide triphosphates (NTPs) may be synthesized with modifications at the base, sugar, or triphosphate chain. Historically, modification of the triphosphate chain has been mainly used to study enzymatic pathways, which results in hydrolysis and transfer of the phosphate from NTP to another molecule. Modification of sugar and base has served a number of different purposes, from pharmaceutical to diagnostic applications.
In many examples of deoxyribonucleotide triphosphate (dNTP) analogs, the original P1—O—CH2(5′) fragment has been modified. One common type of modification is the substitution of an oxygen atom with S, NH, or CR2 (where R is H, an alkyl, or an aryl group and their derivatives). Interest in 5′-NH2-dNTPs (i.e. 5′ phosphoramidates and analogs), in particular, has increased due to their potential utility in genomic analysis (see, e.g., Shchepinov et al., Matrix-induced fragmentation of P3′-N5′ phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms. Nuc. Acids Res. v. 30(17) pp. 3739-3747 (2002)) and DNA sequencing (see, e.g., U.S. Pat. No. 8,324,360 to Kokoris et al.). Certain useful features of 5′ phosphoramidate analogs include their ability to exist in a triphosphate form, which can be utilized by many polymerases (see, e.g., Letsinger et al., Incorporation of 5′-amino-5′-deoxythymidine 5′ phosphate in polynucleotides by use of DNA polymerase I and a phiX174 DNA template. Biochemistry v. 15 pp. 2810-2816 (1975)), and the ability to selectively cleave the P—N bond under acidic conditions (see, e.g., Letsinger et al., Enzymatic synthesis of polydeoxyribonucleotides possessing internucleotide phosphoramidate bonds. J. Am. Chem. Soc. v. 94 pp. 292-293 (1972)).
Modification of the dNTP alpha phosphate, in turn, has been exploited to introduce diverse functional properties, such as attachment points for detectable labels, solid state matrices, and other useful substituents. Examples of nucleotide analogs modified on the phosphate residue and various processes for producing such analogs have been described in several reviews. See, e.g., Koukhareva, Vaghefi and Lebedev, Nucleoside Triphosphates and their Analogs (2005) Chapter 2, “Synthesis and properties of NTP analogs with modified Triphosphate side chains”, Ed. M. Vaghefi, CRC Press, Taylor & Francis, Boca Raton. Triphosphates are of particular importance as substrates for DNA or RNA polymerase that incorporate the nucleotide analogs into long chain nucleic acids. Generally, triphosphates are synthesized by first preparing the nucleoside monophosphates, which are subsequently converted into triphosphates enzymatically, for example, by kinases. However, nucleotide monophosphate (NMP) analogs may not be suitable substrates for kinase enzymes, and the preparation of such specific analogs thus will likely require unique chemical routes.
Though a variety of different analogs are available that mimic nucleotides and their polymers for diverse applications, there remains a need in the art for the development of novel analogs that offer unique combinations of individual properties while retaining the ability to be recognized and acted upon by enzymes. For example, the concept of “sequencing by expansion” has been described, in which a template nucleic acid is converted into an expandable daughter-strand polymeric “surrogate” through template-directed enzymatic synthesis. In one embodiment, the synthesis reaction incorporates dNTP analogs, referred to as “XNTPs” (see, e.g. Kokoris et al., U.S. Pat. No. 8,324,360). Once incorporated into the surrogate, cleavage of the selectively cleavable bonds can effectively expand the polymer, thus increasing the spatial resolution of the individual nucleotides. Such expanded nucleic acid molecules show great promise in, e.g., nanopore-based sequencing systems. For this particular application, it would be advantageous to provide improved polymerase substrate analogs that feature both a selectively cleavable bond and an attachment point for a bulky substituent, such that these features are introduced into the expandable surrogate daughter-strand product.
Thus, one technical object forming the basis of the present invention is to provide improved nucleotide phosphoramidate analogs that are further modified on the alpha-phosphate to enable attachment of a variety of application-specific substituents (e.g. tether molecules) and, furthermore, to provide reliable processes for the synthesis of such novel nucleotide analogs.
All of the subject matter discussed in the Background section is not necessarily prior art and should not be assumed to be prior art merely as a result of its discussion in the Background section. Along these lines, any recognition of problems in the prior art discussed in the Background section or associated with such subject matter should not be treated as prior art unless expressly stated to be prior art. Instead, the discussion of any subject matter in the Background section should be treated as part of the inventor's approach to the particular problem, which in and of itself may also be inventive.